Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most toxic known protein and the causative agent of human botulism. BoNTs have similar structures and functions, comprising three functional domains: catalytic domain (L), translocation domain (HN), and receptor-binding domain (Hc). In the present study, BoNT/E was selected as a model toxin to further explore the immunological significance of each domain. The EL-HN fragment (L and HN domains of BoNT/E) retained the enzymatic activity without in vivo neurotoxicity. Extensive investigations showed EL-HN functional fragment had the highest protective efficacy and contained some functional neutralizing epitopes. Further experiments demonstrated the EL-HN provided a superior protective effect compared with the EHc or EHc and EL-HN combination. Thus, the EL-HN played an important role in immune protection against BoNT/E and could provide an excellent platform for the design of botulinum vaccines and neutralizing antibodies. The EL-HN has the potential to replace EHc or toxoid as the optimal immunogen for the botulinum vaccine. Clostridium botulinum in an anaerobic environment [Botulinum neurotoxin (BoNT) is a neurotoxin produced by ironment ,2. BoNT ironment ,4. Novelironment ,5.BoNT/A–G have similar structures and functions, with amino acid sequence homology of 37–70% . BoNT coThe intoxication process is divided into three steps. First, the Hc domain of BoNT binds to the neuronal membrane on the presynaptic side of the peripheral synapse . Then, tVaccination is an effective strategy to prevent botulism. An investigational pentavalent vaccine  has been the most used human botulism vaccine, which induces neutralizing antibodies against BoNT serotypes A, B, C, D, and E. However, the availability of this vaccine is limited because it is expensive, difficult to produce, and involves a dangerous detoxification process . TherefoAmong the various serotypes of BoNTs, serotype A has attracted the most attention from researchers in various countries, while fewer studies have investigated serotype E. In terms of molecular structure, the L and Hc domains of BoNT/E are located on the same side of the HN domain. It has a unique domain arrangement, which was suggested to be linked to the faster onset of action, making the symptoms of poisoning appear more rapidly, with an impact on human health that cannot be underestimated . BotulisIn the present study, BoNT/E was selected as a model toxin to further explore the immunological significance of each domain. We prepared a series of recombinant antigens derived from the different domains of BoNT/E and systematically explored their immunological characteristics and immunoprotective efficacies. Our results showed that the recombinant EL-HN fragment (L and HN domains of BoNT/E), which was described previously by Shone et al. , has natE. coli (BL21), and the purified proteins were identified by both their molecular weight and reaction with specific antibodies to BoNT/E in immunoblots. SDS-PAGE analysis  with low levels of serum neutralization antibodies. After the third immunization, the protective potency of the above antigens was moderately enhanced. However, the EHc-N antigen did not produce immune protection in mice after three immunizations, and the serum neutralization antibody titer was less than the low measured value of 0.25 IU/mL.In all immunological experiments, the un-nicked EL-HN  was used to immunize mice. The survival rate of toxin challenge and the level of neutralization titer were used to evaluate the immunoprotective potency of the recombinant antigens. As demonstrated in The neutralizing antibody response produced by the combination antigens of EHc-N + EHc-C was also lower than that produced by EHc alone . We specELISA was used to detect antibody titer in mouse sera after immunization with the recombinant antigens. Each recombinant antigen induced a strong and specific antibody reaction against itself, and the antibody titer after the three vaccinations were significantly enhanced . These rp < 0.05), which might be related to the structure of the antigen and the immunity of the combined antigen. It also led to a protective effect that was lower than that of the single antigen.The antibody titer in mice induced by the combined antigen were detected using plates coated with the single component of the combined antigen. According to the results, the antibody response induced by EHc + EHN or EHc + EL-HN was weaker than that of a single antigen . Only immunization with ≥1 μg of EHc could provide 100% protection . In summary, two immunizations with the EL-HN antigen at a low dose provided complete protection for mice challenged with a high dose of BoNT/E.Furthermore, the antibody titer in mouse sera after two immunizations with different dosages of recombinant antigens EHc and/or EL-HN were determined . The levBoNT has been classified as an important biological threat because of its high toxicity and lethality . BotulisE.coli-expressed AL-HN protein without formaldehyde treatment displayed a stable structure and antigenicity. This AL-HN antigen, containing important neutralizing epitopes, produced immune protection, although its protective potency was weaker than that of AHc. Moreover, we found that another antigen BL-HN derived from BoNT/B could provide effective protection; however, its protective potency was also weaker than that of BHc [As another novel research direction, chimeric or fusion molecules with two domains of BoNTs have promising research value . Protectt of BHc .In the present study, we explored the immunological characterizations and immunoprotective efficacy of different functional fragments derived from BoNT/E. Notably, we found that the protective potency of EL-HN was significantly stronger than that of EHc or the other fragments, which was contrasted with the results for these L-HN fragments of BoNT/A and B. Immunization of EL-HN induced very high levels of serum neutralizing antibodies in mice and two immunizations with a low dosage of the EL-HN antigen can induce high levels of neutralizing antibodies. In addition, the protective effect of the EL + EHN combination antigen was stronger than that of EL or EHN alone, indicating that the combination of the two domains had a certain synergistic effect. However, the protective potency of the combination is lower than this EL-HN fragment. Therefore, our data indicated that the EL and EHN domains contain functional neutralizing epitopes and the functional EL-HN fragment is also an important protective antigen. More importantly, these results indicate that there are more effective neutralizing antibody epitopes on EL-HN, especially at the junction of EL and EHN, than on other fragments of BoNT/E.E.coli-expressed EL-HN fragment may retain the enzymatic activity, which supports that functional neutralizing epitopes exist in the EL-HN fragment. The un-nicked EL-HN fragment was expressed as the sole SC protein and was observed to be in DC form after nicking by trypsin. The functional EL-HN fragment is catalytically active proteins and efficiently cleaves their substrate SNAP-25 as active BoNT/E.Property and activity of EL-HN fragment were also defined in this study. Our results indicate that the E. coli was used to express the L-HN protein, which retains the functionality of the parent holotoxin but lacks the neuron-specific targeting contributed by the binding domains in the full-length BoNT, and retained its stable property, activity, and antigenicity. However, using BoNT/E as a model toxin, we found that the protective potency of EL-HN was significantly stronger than that of other fragment antigens, including EHc. These results are the first to reveal the strong protective effect of the L-HN fragment from BoNT/E.In general, the BoNT antigenic epitopes are distributed widely among the full toxin, and the core immunogen for immunoprotection comprises the functional neutralizing epitopes. The L-HN fragment derived from BoNTs represents the N-terminal two-thirds of the neurotoxin. In the present study, Among the neutralizing antibodies against BoNT/E, many target the L  and HN  domains Generally, toxin-protective antibodies should have a neutralization capacity. The mechanism of action of neutralization antibodies is to inhibit the binding of the toxin to the neuronal cell surface receptor and clear the toxin in circulation ,44. In t50 of 223 ng. Furthermore, catalytically inactive Clostridium botulinum holoproteins (ciBoNT HPs) might provide a stronger immunological response that elicits relatively higher potency than the Hc-based BoNT/E vaccines. In the present study, EL-HN delivered via the two-vaccination approach resulted in an ED50 of 17 ng, which was similar to the potency of ciBoNT/E HP (ED50 = 12 ng). Therefore, the potency of EL-HN as a subunit vaccine is equivalent to holoproteins of BoNT/E. Przedpelski et al. [W and LCHCN are more potent vaccines than HCC. LCHCN had a similar vaccine potency as M-BoNT/A1W. M-BoNT/A1W elicited a common dominant antibody response to LCHCN, but a varied HCC antibody response in mice. This may be a general concept applicable to other BoNTs. The full-length not toxic BoNTs or other derivations represent a novel strategy for the development of vaccines against botulism.In this study, the serum antibodies of EL-HN-immunized mice had stronger neutralizing activity than those of EHc and other fragments, which indicated that EL-HN contains more or important functional neutralization epitopes that play an important role in immune protection against BoNT/E. Webb et al.  demonstri et al.  also demOur data strongly indicated that the EL-HN region is important to generate protective antibodies and should aid the design of BoNT/E vaccines. With regard to BoNT/E, EL-HN is more suitable as a subunit vaccine of BoNT/E than EHc or other antigens. In addition, it is interesting that the recombinant EL-HN functional fragment is used to screen for L-HN-specific, but not Hc-specific, neutralizing antibodies to treat botulism.The functional EL-HN fragment had the highest protective efficacy and contained some functional neutralizing epitopes. Furthermore, the EL-HN retained the property and activity of the toxin. Thus, the EL-HN has the potential to replace EHc or toxoid as the optimal immunogen for the botulinum vaccine. The functional L-HN fragment provides an additional tool that allows us to understand the structure–function relationship of the different domains of BoNT in the process of intoxication or degree of immune protection. Further biological function and immunological characterizations of the L-HN fragment will be performed in future studies. These findings offer a new strategy to develop vaccines and therapeutic antibodies against BoNTs.Female Balb/C and KM mice were obtained from Vital River Laboratory Animal Technology Co. Ltd. . The mice were housed under pathogen-free conditions  and randomly assigned to different groups. All animal experiments were performed with the permission of the Institute of Animal Care and Use Committee (IACUC) at the Academy of Military Medical Science, and the ethical approval number was IACUC-DWZX-2019-017.Escherichia coli, gene fragments of each function fragment . A single colony was inoculated in a 2×YT medium containing 100 μg/mL ampicillin and cultured at 37 °C with shaking at 220 rpm until the absorbance at 600 nm (OD600) of 1.0 was reached. The cells were inoculated into 400 mL of 2×YT medium (containing ampicillin at 100 µg/mL) and cultured at 37 °C with shaking at 220 rpm until OD600 of 0.8 was reached. Then, isopropyl-β-D-thiogalactopyranoside  was added to the cell culture such that its final concentration was 0.2~0.4 mM to induce the expression of recombinant antigens. The cells were cultured overnight at 16 °C and centrifuged at 6000× g for 30 min to collect the cells. The cells were then resuspended in a binding buffer  and lysed using an ultrasonic homogenizer in an ice water bath. The cells were centrifuged at 12,000× g for 20 min at 4 °C to collect the supernatant.Each recombinant plasmid was transformed into the An AKTA Explorer  was used for chromatographic purification of these recombinant proteins with His-tag at the C-terminus from the supernatant. The filtered lysates were then loaded onto a HisTrap HP  column that was pre-equilibrated with 10 column volumes (CV) of binding buffer. Gradient elution with elution buffer was used to elute the recombinant protein from the column. The eluted sample was collected, and the buffer of the final product was exchanged with phosphate-buffered saline . The purity and specificity of the recombinant antigens were checked using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.The antigenicity of the recombinant protein was detected by Western blotting. After separation by 10% SDS-PAGE, the EHc, EHc-N, EHc-C, EL-HN, EHN, and EL proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using a film transfer apparatus and blocked with 5% skim milk at 37 °C for 1 h. The membrane was then incubated with 1:5000 diluted antibodies from hyperimmune anti-BoNT/E horse sera overnight at 4 °C. After washing with TBST , the membrane was incubated with 1:2000 diluted goat anti-horse IgG-HRP  at 37 °C for another 1 h. After washing with TBST, Western blotting solution  was added to the membrane, and it was exposed on a gel imager .The recombinant EL-HN fragment was proteolytically nicked by trypsin  (1:100 ratio of trypsin to EL-HN) for 30 min at 35 °C. Next, 4 μg/mL trypsin inhibitor  was added to stop the digestion. Both the un-nicked EL-HN  and nicked EL-HN  were determined by SDS-PAGE.A recombinant plasmid pET32a-SNAP-25 encoding human SNAP-25 protein (aa 1-206) with his tag was constructed in our Lab. The SNAP-25 protein was expressed in BL21(DE3) cells and purified by affinity chromatography. The proteolytic activity of EL-HN was assayed in vitro using the SNAP-25 protein. The assay was performed in a final volume of 50 µL , containing different concentrations of EL-HN (from 2.56 nmol to 1600 nmol) or BoNT/E (from 4.8 nmol to 600 nmol and from 2 μmol or 5 μmol substrate SNAP-25). The amounts of cleavage of SNAP-25 (2 or 5 μmol/L) by the EL-HN or BoNT/E were determined by incubating the samples at 37 °C for 30 min. The reactions were stopped by adding the 4× concentrated SDS-PAGE sample buffers. The extent of cleavage was then evaluated following electrophoresis on SDS-PAGE gels.50 assay. Groups of four mice were used for each concentration, and the un-nicked and nicked EL-HN were diluted in sterile normal saline. The lowest amount of toxin that killed 50% of mice within four days was considered to be one minimal lethal dose (LD50) and was expressed as the number of LD50 units/mg of protein.The neurotoxicity of the un-nicked and nicked EL-HN in mice following an intraperitoneal injection as a full neurotoxin was determined using an LDn = 10 per group), with a PBS group serving as the control. Single antigen (10 μg) and combination antigens (5 μg each) were diluted with sterile PBS and formulated with 0.2% (w/w) aluminum hydroxide gel . The mice were vaccinated intramuscularly with 100 μL of subunit vaccine preparation. The injection protocol was a total of three immunizations with an interval of 3 weeks. Then, 100~200 μL blood from each mouse was collected at 18 days after two or three vaccinations. After 2 or 3 immunizations, the 102, 103, or 104 LD50  BoNT/E was used to challenge the mice, and the survival rate was calculated for one week.We divided 6–8-week-old specific pathogen-free (SPF) female Balb/c mice into groups , which is assayed using botulinum antitoxin standard , 3 weeks after the last vaccination. The survival rate was observed once per day for a week.A dose-dependent protective experiment was carried out on the recombinant antigens with a better protective effect. The female Balb/c mice were immunized with once or twice using serially diluted  antigen and the negative control; 100~200 μL blood from each mouse was collected at 18 days after one or two vaccinations. These immunized mice were challenged with 10v/v) Tween-20) for 3 times. Mouse serum samples were 4-fold serially diluted from 1:100 using a 2% skim milk blocking solution. The diluted samples (100 µL) were added to each well and incubated for another 90 min at 37 °C. After washing with PBST six times, the plates were added with 100 µL of 1:5000 diluted IgG-HRP per well and incubated for 30 min at 37 °C. The samples were washed with PBST, incubated for 10 min at 37 °C, followed by adding 50 µL of citrate buffer (pH 5.0) with 0.02% (v/v) hydrogen peroxide and 0.04% (w/v) o-phenylenediamine to each well. Then, 2 M H2SO4 at 50 µL per well was used to stop the reaction, and the absorbance was read at 492 nm on a microplate reader. Absorbance values at 492 nm (OD492) that were greater than 0.5 and ratios of the absorption value of the experimental group and the negative control group greater than 2.1 were regarded as positive results. Serum samples taken from individual mice in each group were measured separately, and the geometric mean titer (GMT) for each group was calculated using the average values.Humoral immune responses were measured using an enzyme-linked immunosorbent assay (ELISA). Each well of a 96-well enzyme-linked plate was coated overnight at 4 °C with 100 μL of recombinant antigen (2 µg/mL). The plates were incubated with 200 μL of 2% skim milk blocking solution at 37 °C for 2 h and then washed three times with PBST . Dilution buffer  was then used to adjust the total volume to 2.5 mL before incubation at room temperature for 1 h to fully react the neurotoxin and antibodies. Female KM mice weighing 15~18 g were then injected with the mixtures intraperitoneally using 500 µL per mouse . Starting at 12 h after injection, the survival of the mice was observed for a week, once every day. The serum neutralizing antibody titer were calculated as international units per milliliter (IU/mL) relative to the World Health Organization (WHO) BoNT/E antitoxin levels.A BoNT/E neutralization assay  was usedt-test was used to analyze differences in the antibody immune responses between the groups. To ascertain the statistical differences in survival between the treatment groups, Fisher’s exact test was used. Statistical significance was accepted at p values < 0.05 for the data from all tests. Probit analysis at the 95% confidence level was used to determine the effective dose protecting half of the mice (ED50) using IBM SPSS Statistics .One-way analysis of variance (ANOVA) or using Student’s


Kerley A-lines are generally apparent in patients with pulmonary edema or lymphangitic carcinomatosis. There are two main thoughts regarding the etiology of Kerley A-lines, but no general agreement. Specifically, the lines are caused by thickened interlobular septa or dilated anastomotic lymphatics. Our purpose was to determine the anatomic structure represented as Kerley A-lines using 3D-CT lung segmentation analysis.We reviewed 139 charts of patients with lymphangitic carcinomatosis of the lung who had CT and X-ray exams with a maximum interval of 7 days. The presence of Kerley A-lines on X-ray was assessed by a radiologist. The A-lines on X-ray were defined as follows: dense; fine (< 1 mm thick); ≥ 2 cm in length, radiating from the hilum; no bifurcation; and not adjacent to the pleura. For cases with Kerley A-lines on X-ray, three radiologists agreed that the lines on CT corresponded with Kerley A-lines. The incidence of A-lines and the characteristics of the lines were investigated. The septal lines between lung segments were identified using a 3D-CT lung segmentation analysis workstation. The percentage of agreement between the A-lines on CT and lung segmental lines was assessed.On chest X-ray, 37 Kerley A-lines  were identified in the 22 cases (16%). Of these, 4 lungs with 12 lines were excluded from analysis due to technical reasons. Nineteen of the 25 lines (76%) corresponded to the septal lines on CT. Of these, 11 lines matched with automatically segmented lines  by the workstation. Two lines (8%) represented fissures. Four lines corresponded to the bronchial wall/artery  or vein .Kerley A-lines primarily represented thickened and continued interlobular septal lines that corresponded to the septa between lung segments and subsegments.The online version contains supplementary material available at 10.1007/s11604-021-01215-4. In 1951, Kerley categorized linear shadows on chest X-ray as Kerley A-, B-, and C-lines . Kerley The Kerley A-line is a longer linear shadow in the upper lobes that is oriented from the hilum toward the pleura . The KerIn an effort to settle this difference of opinion regarding the Kerley A-line, we used a sophisticated 3- dimensional CT imaging technique. At the height of the research fervor involving the Kerley A-line, high-resolution CT was not available, thus pulmonary lymphangiography and pathologic specimens were used to address this question. Currently, we have high-resolution CT technology that enables us to obtain MPR images and 3D anatomic images, which together will contribute to revealing the identity of the Kerley A-line which has been a matter of debate for 70 years. We speculate that the A-line represents thickened septal plates between lung segments and intersegmental septa based on our daily clinical experience in interpreting chest CT scans of patients with pulmonary edema and lymphangitic carcinomatosis. Indeed, we support the “thickened lung connective tissue septum” proposed by Heitzman and Grainger. The purpose of this study was to determine whether the Kerley A-line consists of lung intersegmental septa using 3D-CT lung segmentation analysis.This retrospective, cross-sectional study was approved by our institution’s Ethics Committee for Clinical Research with a waiver of informed consent. We studied consecutive patients who underwent chest CT with a suspected diagnosis of lymphangitic carcinomatosis from January 2009 to January 2019. An initial listing of the patient exams for this study was obtained through a search of the radiology picture archiving and communication system (PACS) and radiology information system (RIS). The final diagnosis of lymphangitic carcinomatosis was based on chest CT findings and clinical course. All chest CT exams were reported by board-certified radiologists and re-reviewed by a thoracic radiologist with 11 years of experience (N.T.) to confirm the diagnosis of lymphangitic carcinomatosis. All patients with a final diagnosis of lymphangitic carcinomatosis and who had chest CT and X-ray studies within 7 days were included in this analysis ; 2.8mAs; 1.2 mm focal spot; 10:1 grid; and 52 lines/cm . Mobile X-rays were obtained with the following parameters: 90 kVp; 25 mA; 49 ms; 1.2mAs; and 6:1 virtual grid .CT examinations were performed using two different scanners: a 64-row LightSpeed VCT scanner  and a 320-row Aquilion One scanner . CT scanning was performed during a breath-hold in full inspiration with the patient in the supine position. Scanning parameters were as follows: voltage, 120 kVp; current, AEC; collimation, 0.5 (One) or 0.625 mm (VCT); rotation time, 0.5 s; matrix, 512 × 512; and slice thickness, 1 (One) or 1.25 mm (VCT).One thoracic radiologist (S.M. with 40 years of experience) blinded to the CT images reviewed the chest X-ray images and the presence or absence of the Kerley A-lines was determined. The A-lines on chest X-ray were defined based on the classic Kerley definition as follows: dense; fine (< 1 mm thick); ≥ 2 cm in length radiating from the hilum; no bifurcation; and not adjacent to the pleura . The length of the A-line was measured. Another thoracic radiologist (N.T. with 11 years of experience) blinded to the chest X-ray images reviewed the CT images and the presence or absence of the thickened septal lines were determined.Next, three thoracic radiologists  compared the chest X-ray and CT images of the cases with Kerley A-lines independently to detect the corresponding anatomic structure of the Kerley A-lines. Discrepancies were resolved by consensus. CT images with a slice thickness < 1.5 mm were reviewed at a workstation . Any user-defined reconstructions were available for review, including axial images, multiplanar reformatted images (MPRs), maximum intensity projection (MIP), and average intensity projection (AIP).3D-CT lung segmentation analysis was performed using a commercially available workstation . Lung segmentation was based on bronchial segmentation. For bronchial segmentation, the fully-automated workstation extracted bronchial trees and a thoracic radiologist (N.T. with 11 years of experience) named each bronchus at the level of the fourth bronchial generation based on the definition described by Yamashita Table .Table 1NBetween January 2009 and January 2019, there were 379 chest CT studies (207 patients) with a suspected diagnosis of lymphangitic carcinomatosis. Of these studies, 169 patients were ultimately diagnosed with lymphangitic carcinomatosis. There were 139 patients who had a chest CT and chest X-ray within a 7-day interval.On chest X-ray, 37 Kerley A-lines  were identified in the 22 patients (16%). On chest CT, there were 117 patients (84%) with thickened septal lines. Twenty of 22 patients (91%) had both Kerley A-lines on chest X-ray and thickened septal lines on chest CT . Twenty-five Kerley A-lines  were analyzed by the radiologists and the lung segmentation software to identify the corresponding anatomic structures on chest CT. The characteristics of Kerley A-lines are summarized in Tables In this study, we showed that most Kerley A-lines consisted of lung intersegmental/subsegmental septa in lymphangitic carcinomatosis patients using 3D-CT lung segmentation analysis. The presence of intersubsegmental septa has not been confirmed in the literature, but the results of this study support the presence of intersubsegmental septa.A Text-book of X-ray Diagnosis [In 1951 Kerley definedlonger linear shadows in the upper lobes, oriented from the hilum toward the pleural as Kerley A-lines in the 2nd edition of the book entitled iagnosis , 8. Nagaiagnosis . In thisiagnosis . Our stuThe A-line on chest X-ray radiated from the hilum; however, many A-lines on CT did not continue to the hilum. The A-lines on chest X-ray were not adjacent to the pleura; however, many A-lines on CT scans were adjacent to the pleura. The reason for these findings was that the septal line between the upper lobe segment, which corresponded to the Kerley A-line, ran straight and extended obliquely and medially from the anterior chest wall on the anteromedial lung portion. Berkman et al.  comparedThe incidence of Kerley A-lines on chest X-ray in patients with lymphangitic carcinomatosis was 16%. The incidence of thickened septal lines on CT in patients with lymphangitic carcinomatosis was 84%. Even if the septal line was visible on CT, it was not always possible to visualize the A line on chest X-ray. Whether the A line was or was not noted was due to the variation between septal lines (variation in lung segmentation). The lung segment septum was not constant across all segments and may or may not be present during the development of the lung. The presence of accessor and incomplete fissures supports this notion. In contrast, even though there was no pulmonary stasis on CT, the A-line on X-ray, a sign of pulmonary congestion, was highlighted in this study. The incidence of these pseudo-A-lines which were formed by the bronchial wall, arteries, fissures, and veins was 9%.Although this study supports the interlobular theory, we do not dismiss the dilated lymphatics theory because lymphatic vessels are one of the structures that comprises the interlobular septum. The historic investigators of the Kerley A-lines also accepted the alternative hypothesis, in part, and advocated their own theories, as we have done. As Grainger concluded , it is mThis study had several limitations. First, there were differences in examination dates, position (standing or supine and hands up or down) and the level of inspiration between chest X-ray and CT. Second, lung segment nomenclature is not perfect because there are normal variations in the bronchial bifurcation. In this study, we used lung segmentation based on the bronchial bifurcation because most of the available CT scans were non-contrast CT scans. The results may differ with lung segmentation based on vascular bifurcation. Third, we did not include short lines oriented at the hilum (< 2 cm), which is more frequently experienced clinically as A-lines. These lines are likely to be the short interlobular septum, rather than the intersegmental septa . WhetherIn conclusion, Kerley A-lines represent thickened and continued interlobular septal lines that correspond to the septal plate between lung segments or subsegments. The bronchial wall, arteries, fissures, and veins may be mistaken for a pseudo-A-line with a 9% incidence when defining a true A-line as a thickened lung segment septum. In this study, to celebrate the 70th anniversary of the Kerley A-line report, we assessed the validity of the Kerley A-line in making a final decision.Supplementary file1 (PDF 3747 KB)Below is the link to the electronic supplementary material.


Defects and in-plane strain have significant effects on the electronic properties of two-dimensional nanostructures. However, due to the influence of substrate and environmental conditions, defects and strain are inevitable during the growth or processing. In this study, hybrid density functional theory was employed to systematically investigate the electronic properties of boron-phosphide monolayers tuned by the in-plane biaxial strain and defects. Four types of defects were considered: B-vacancy (B_v), P-vacancy (P_v), double vacancy (D_v), and Stone–Wales (S-W). Charge density difference and Bader charge analysis were performed to characterize the structural properties of defective monolayers. All of these defects could result in the boron-phosphide monolayer being much softer with anisotropic in-plane Young’s modulus, which is different from the isotropic modulus of the pure layer. The calculated electronic structures show that the P_v, D_v, and S-W defective monolayers are indirect band gap semiconductors, while the B_v defective system is metallic, which is different from the direct band gap of the pure boron-phosphide monolayer. In addition, the in-plane biaxial strain can monotonically tune the band gap of the boron-phosphide monolayer. The band gap increases with the increasing tension strain, while it decreases as the compression strain increases. Our results suggest that the defects and in-plane strain are effective for tuning the electronic properties of the boron-phosphide monolayer, which could motivate further studies to exploit the promising application in electronics and optoelectronics based on the boron-phosphide monolayer. Two-dimensional 2D) nanostructures with an atomic thickness have attracted an increasing amount of attention due to their large surface-to-volume ratio and unique electronic properties. Graphene, a representative for 2D nanostructures, was first prepared by mechanical exfoliation (repeated peeling) of highly oriented pyrolytic graphite in 2004 . Due to D nanostrAmong the hexagonal honeycomb nanostructures, the boron-phosphide monolayer is a typical binary compound with good structural and mechanical stability, adjustable polarity of transport, and high carrier mobility ,12,13, aFor the hexagonal boron-phosphide monolayer, what are the effects of defects and strain on the electronic properties? Furthermore, the influence of these factors on the structural and electronic properties plays a key role in the application of nanoelectronic devices. In this study, the first-principles study based on hybrid density functional theory was performed to systematically explore the effects of defects and strain on the structural and electronic properties of the boron-phosphide monolayer. The screened hybrid density functional HSE06 was employed to accurately evaluate the band gaps of the pure and defective boron-phosphide monolayers for exploiting their potential applications in nanoelectronics and optoelectronics.−5 eV, respectively. The integration of the Brillouin zone was sampled by using a Gamma-centered k-point grid. The selection of k-points was optimized with the energy error of the system smaller than 1 meV/BP. All of the studied boron-phosphide monolayers, including the pure, defective, and strained ones, were modeled in the x–y plane. For the pure and strained layers, a hexagonal primitive cell was adopted. Thus, the optimized k-points grid was 17 × 17 × 1. For the defective systems, an orthogonal 4 × 3 supercell containing 4 × 4 primitive cells was used, and the optimized k-points grid was 5 × 5 × 1. To eliminate the interactions between neighbor layers, a vacuum space of 15 Å was applied along the normal direction of the boron-phosphide monolayer. For structural optimizations, the Hellmann–Feynman forces using the conjugate gradient algorithm were calculated within a force convergence of 0.01 eV/Å [Our calculations were performed using the first-principles study based on spin-polarized density functional theory (DFT) within the projector augmented plane wave method, as implemented in Vienna ab initio simulation package (VASP) ,28. The .01 eV/Å . The scr.01 eV/Å , which wa = b = 3.21 Å, and the B-P bond length is 1.86 Å, which are well consistent with previous studies [zp orbitals of the B and P atoms are critical to the direct band gap feature. For the xp, yp, and s orbitals, a strong σ bond is formed between the B and P atoms, which is located in the deep energy level and plays a key role in maintaining the structural stability of the boron-phosphide monolayer.The structural and electronic properties of pure boron-phosphide monolayer are investigated firstly by comparing them with the defective systems. The optimized structure is shown in  studies ,14,15. I studies ,13,14,15lP-P = 2.22~2.26 Å) [lB-B = 1.61~1.87 Å) [e in the pure monolayer, indicating weak ion bond interactions between the B and P atoms. For the case of the D_v defect displayed in The optimized geometrical structures of defective boron-phosphide monolayers are shown in ~2.26 Å) ,34 and b~1.87 Å) ,36. BadeC11, C22, C12, and C44, in-plane Young’s modulus along an arbitrary direction θ (θ is the angle relative to the positive x-direction of the sheet) can be expressed as [c = cos(θ) and s = sin(θ). The polar diagrams of C(θ) for pure and defective boron-phosphide monolayers are displayed in To assess the structural stability of these defective monolayers, the formation energy is calculated as essed as C(θ)=C11zp orbital of P atom, and a small part comes from the zp orbital of B atom, forming the hybrid interaction of π bond between the B and P atoms. In addition, these zp orbitals are the main carriers and spread nearly in the whole surface areas, exhibiting good electronic conductivity. As shown in B. Both the spin-up and spin-down channels exhibit indirect band gap features. The corresponding band gaps are 0.24 and 0.54 eV predicted using PBE functional. By employing the screened hybrid density functional HSE06, much larger band gaps of 0.93 and 1.03 eV are obtained for the spin-up and spin-down channels, respectively. Here, it should be pointed out that the HSE06 functional has a great correction on the spin-up band structure, not only on the band gaps. For example, the PBE functional predicts that the VBM of the spin-up band structure is located at the Γ point, whereas the HSE06 functional reveals it is located at the X point. Although there are some differences in the band structure calculations, it does not affect the indirect band gap semiconducting properties and the total magnetic moment. In view of spin-charge density shown in xp and yp orbitals for the double coordinated B atom. The small contribution comes from the xp and yp orbitals of the adjacent two P atoms.Next, we calculate the electronic property of the boron-phosphide monolayer tuned by the B_v, P_v, D_v, and S-W defects. ε = (a − a0)/a0, where a and a0 are the lattice constants for strained and equilibrium states, respectively. ε. In the range of −5 to 5%, both the PBE and HSE06 functionals predict a similar tendency of band gap. With the increasing tension strain, the band gap increases, while it decreases as the compression strain increases. The results of HSE06 functional calculations display that the band gap can be tuned from 1.07 to 1.55 eV in the range of ε = −5~5% with an average slope of 0.05 eV per strain, offering an effective method to tune the band gap of the boron-phosphide monolayer.For 2D nanostructures, the strain plays a key role in their band gap engineering. Previous studies have confirmed that the in-plane strain is very effective for regulating the electronic band gaps of 2D monolayers. Herein, the in-plane strain is defined according to the following formula: In summary, spin-polarized density functional theory calculations are employed to explore the effects of B_v, P_v, D_v, and S-W defects and in-plane biaxial strain on the structures and electronic properties of the boron-phosphide monolayer. The main results are as follows: (a) All of the defects can induce local atomic reconstructions in the boron-phosphide monolayer. (b) All of the defects can not only result in low in-plane Young’s modulus but also induce anisotropic elastic properties in the boron-phosphide monolayer. (c) Their effects on electronic properties are significant. The B_v defect can result in metallic property; the P_vacancy defect leads to magnetic semiconducting with indirect band gaps for spin-up and down-down channels. The D_v and S-W defects can induce indirect band gap semiconducting. The band gaps of these defective monolayers are ~ 1.0 eV, which is comparable to that of silicon. (d) The in-plane biaxial strain can monotonically tune the band gap of the boron-phosphide monolayer with an average slope of 0.05 eV per strain. The band gap increases with the increasing tension strain, while it decreases as the compression strain increases. The above results suggest that defects and in-plane strain are effective in adjusting the electronic properties of the boron-phosphide monolayer, which can promote further research and development in electronics and optoelectronics based on the boron-phosphide monolayer.


Buckwheat by-products may be used as promising food ingredients due to their nutritional composition. Buckwheat husk (BH) may be used in meat products as a source of valuable compounds. In this study, the addition of BH to the quality of frankfurter-type sausages was investigated, aiming to reduce buckwheat waste and to develop nutritionally enriched sausages. For the purpose of this study, a range of measurements, as well as observations, have been carried out. This included the following: pH, weight losses, yield, the instrumental color and texture measurement, protein digestibility, polyphenols, amino acid, trace elements analysis, and the organoleptic evaluation. Compared with no BH sausages, the cooking losses of sausages with 3% BH were higher, while storage losses were lower. BH increased the hardness of sausages after two weeks of storage. The growing addition of BH resulted in a decrease in L* and b*. This change of color resulted in its lower level of consumer acceptability. BH addition did not reduce the protein digestibility. The total amino acid content increased with the increasing husk addition, from 161.8 mg/kg to 228.0 mg/kg. Moreover, BH increased the content of manganese, calcium, potassium and magnesium. This research suggested that incorporation of BH improved the nutritional value of sausages with minimal changes in technological and sensory properties. Tara spinose , cohesiveness, elasticity [mm], chewiness [Nm] and gumminess were evaluated at room temperature (22 ± 1 °C), directly after the production process and after 2 weeks of cold storage.Y  =  93.8; x  =  0.313; y  =  0.319).The instrumental color measurement of sausages was performed on the day of production and after two weeks of cold storage. Sausages were cut crosswise into 10–15 mm thick slices. Color of the surface was measured using a Konica Minolta Chroma Meters CR-400  and was expressed by L (lightness), a (redness), and b (yellowness) parameters in CIE Lab system. The chroma meter was set at D65 illuminant and 10° standard observer and calibrated before each measurement against a standard white tile  marked with 4-character code were subjected to the evaluation at room temperature (22 ± 1 °C), under white light. The evaluation was carried out by the scaling method according to a 9-point acceptance scale, where 1 meant “extremely dislike” and 9 meant “extremely like”. A ranking assessment in which evaluators were asked to rank the samples from least to most preferred was also conducted.v/v, Sigma-Aldrich, Steinheim, Germany). For the identification of polyphenols, the protocol proposed by Kolniak-Ostek [v/v (solvent A) and 100% acetonitrile (solvent B). The single components of polyphenols were characterized based on the retention time and the accurate molecular masses. The data obtained from UPLC-MS were analyzed in the MassLynx 4.0 ChromaLynx . Phenolic acids were monitored at 320 nm, flavanols at 280 nm, flavonols at 360 nm and flavones at 340. The PDA spectra were measured over the wavelength range of 200–600 nm in steps of 2 nm. The retention times and spectra were compared to those of the authentic standards .The extraction of polyphenols was determined using the method described by Püssa et al.  and prevak-Ostek  was usedThe amino acid composition of samples was determined by ion-exchange chromatography after 23 h hydrolysis with 6 N HCl at 110 °C. After cooling, filtering and washing, the hydrolyzed sample was evaporated in a vacuum evaporator at a temperature below 50 °C. The dry residue was dissolved in a buffer of pH 2.2. The prepared sample was analyzed using the ninhydrin method ,37. The 3) and a solution containing pancreatic–intestine extract  and bile salts  was introduced to simulate intestine digestion. This stage was continued for 2 h at 37 °C with shaking. After digestion samples were centrifuged. Non-digested proteins present in the supernatant were precipitated with 10% TCA (trichloroacetic acid). The percentage content of protein nitrogen released into the obtained supernatants was compared to the content of protein in the non-digested samples with the use of the Kjeldahl method [Samples were digested with the in vitro method simulating multienzymatic two-stage  digestion ,39. The l method .3 of concentrated nitric acid (V) (CTC—reagent grade) was added to an aliquot of a homogeneous sample (from 0.1 g to 0.5 g), then the samples were mineralized in the MARS 6 microwave samples preparation system. The mineralisates were quantitatively transferred to 10 cm3 measuring vessels with redistilled water. The mineralization was carried out in accordance with PN-EN 13805:2003 [The samples were mineralized “wet” in a closed microwave system. Following this, 5 cm805:2003 . Atomic 805:2003 . While t805:2003 . The metThe collected data of the two production batches were analyzed statistically with TIBCO Statistica, version 13.3 . Two-factor analysis of variance (ANOVA) and Duncan’s multiple range test allowed for the determination of the statistically significant differences. Results are presented as mean ± SE (standard error). The applied additive in the form of ground buckwheat husk (BH) did not affect the pH value of sausages, which was within the range 6.0–6.2. However, the addition of BH had a significant effect on the weight losses during the thermal treatment and at the same time, on the process yield .The experimental products lost between 14% and 15.5% of their initial weight and the yield of production ranged from 84.5 to 85.8%. The highest weight losses during cooking and the lowest values of yield were observed in sausages with 3% addition of buckwheat husk. On the other hand, during storage, BH effectively reduced weight losses from 1.93% in the control sausages to 1.36% in sausages with its highest content. This indicates that the addition of buckwheat husk has a positive effect on water retention during the storage of frankfurter-type sausages. These results are supported by the study of Sol-Hee Lee et al. , in whicIn our research, the share of buckwheat husk in the composition also resulted in the changes in the texture profile of sausages . At the Caesalpinia sappan L. (CS) extract to cooked pork sausages. In our own research, the growing addition of buckwheat husk also resulted in the decrease in yellowness of frankfurter-type sausages. Saturation of b* also decreased during cold storage. The observed changes in color are probably related to the color of the BH . The variants differed significantly in the values obtained for the measurement of the L* parameter, directly after the production, i.e., along with the increase in the share of buckwheat husk, the sausages were darker. The same results were obtained by Salejda et al. , by addiIn both production batches, the acceptability of color, aroma, taste, consistency and hardness of frankfurter-type sausages were evaluated. The average results of these evaluations are summarized in As shown in Decreased digestibility of foodstuffs produced with the addition of husk ,61,62 reProcessing buckwheat husk into frankfurter-type sausages had a significant effect on mineral content . The addO-alpha-l-rhamnoside, sinapinaldehyde, juniperic acid, isorhamnetin-3-galactoside-6″-rhamnoside, kaempferol-7-O-alpha-l-rhamnoside, sinapinaldehyde, quercetin derivatives, rosmarinic acid, flavonoid derivatives, vitexin (apigenin-8-C-glucoside), apigenin-7-O-glucoside, apigenin, kaempferol-3-glucoside-3″-rhamnoside, vitexin-2″-O-rhamnoside.Sausages based on buckwheat husk can also be an excellent source of phenolic compounds. We found the following components in the studied sausages : (+)-catFagopyrum esculentum. Both, quercetin and vitexin were characterized by antioxidant activity stronger than synthetic antioxidant BHT [Serjania erecta Radlk leaves, protect PC12 cells from Aβ25–35 peptide-induced toxicity [The main phenolic compounds identified in the studied products were vitexin and their derivatives, except in the control. The variant with the highest addition of buckwheat husk was found to contain the highest concentration of those compounds (dant BHT . These fdant BHT , may be toxicity . We concluded that the buckwheat husk can be used as a non-meat additive to increase the nutritional value of frankfurter-type sausages, without compromising their technological quality. Notably, BH sausages appeared to be a good dietary source of essential amino acids, trace elements and phenolic compounds. Further studies should take into account the impact of BH on the microbial quality of sausages, as well as in vivo testing. These studies will allow for the application of BH as a commercial food additive to develop novel functional sausages. The potential consumer should be aware of the beneficial effects of these new products on health and be convinced to consume them, despite differences in color and taste, or consider the modification of recipe or production technology limiting this adverse effect.


Age at KT is associated with the worsening of arterial stiffness in the late post-transplantation period . A high PWV at 1 year was associated with a potential for recovery during follow-up . (4) Conclusions: The absence of PWV worsening in the late post-transplantation period was significantly associated with a lower risk of CV events, whereas early changes in PWV were not. Finding an intervention capable of reducing long-term PWV could improve the prognosis of KT recipients.(1) Background: Increased arterial stiffness is associated with cardiovascular (CV) diseases in end-stage renal disease (ESRD) patients, and CV mortality remains higher in kidney transplantation (KT) recipients compared to in the general population. KT is associated with an improvement in arterial stiffness in the early post-transplant period, followed by a potential re-worsening in the late period. In a cohort of KT patients, we evaluated the associations of pulse-wave velocity (PWV) measured at different time-points  with CV morbi-mortality, as well as the evolution between these measurements with CV morbi-mortality. (2) Methods: Forty KT recipients with a 10-year follow-up were included. The association of PWV with CV events was assessed with multivariable cox analysis. Backward linear regressions were conducted to identify the determinants of PWV at 1 year and those of the long-term evolution of PWV after KT (delta PWV at 1 year—latest PWV). (3) Results: The absence of arterial stiffening during the long-term follow-up after KT is associated with a lower CV outcome rate (HR for the delta PWV = 0.76 (0.58–0.98),  Cardiovascular (CV) diseases are the leading cause of death in patients with end-stage renal disease (ESRD). After kidney transplantation (KT) and the restoration of renal function, the risk of CV mortality remains high compared with the general population . IncreasThe mechanisms underlying arterial stiffness in KT are numerous, involving the pretransplant history of patients . Age also remains a key determinant of PWV in KT . NeverthTherefore, considering the potential improvement of arterial stiffness in the early post-transplantation period ,7—and poA detailed description of the TRANSARTE study has been published previously . Briefly® technique , which simultaneously records arterial pulse waves at carotid and femoral sites. The propagation time (t) of the incident wave was measured through mechanotransducer probes, the carotid–femoral distance (D) was evaluated with a tape measure, and PWV (m/s) was obtained by the formula PWV = D × 0.8/(t).PWV measurements were performed using the Compliorp-values are considered significant if they are <0.05. The association of PWV with CV events was assessed using Cox models. Backward linear regression was performed to identify the determinants of PWV at 1 year, and absolute changes in PWV post-transplantation . Gender, age at transplantation; mean arterial blood pressure (MBP); hypercholesterolemia; diabetes mellitus; smoking; history of angina or myocardial infarction; previous strokes; CNI (Tacrolimus/Cyclosporine); and cause of ESRD were considered as independent variables. In addition, PWV at one-year post-transplantation was considered in the model focusing on late change in PWV. All variables with a p < 0.15 in univariable analysis were used as candidate variables in the multivariable model prior to backward selection procedures.Analyses were performed using R version 4.0.2 . The means are presented with SD and median, with 25th and 75th percentiles. The n = 6) or a deceased (n = 34) donor allograft, the median age at transplantation (years) was 53 [47–60]. 19 (47.5%) were female. Of the subjects, 5 (12.5%) were diabetic, 2 (5.0%) had history of angina or myocardial infarction, and 2 (5.0%) had previously experienced strokes. The mean pre-transplantation, 1-year post-transplantation and latest post-transplantation PWVs (m/s)  were 8.9 ± 2.45, 9.0 ± 2.8, and 8.9 ± 2.4 (m/s), respectively (p = 0.96). The majority of patients received calcineurin inhibitors (CNI) (95%), most of them cyclosporine (55.3%). During the median follow-up period of 10.9 years, 12 patients died, 11 patients had a CV event and 6 patients returned to dialysis ). On the contrary, the evolution of PWV between the pre-transplant period and the early post-transplantation period, as well as the PWV at 1 year, were not significantly associated with CV morbi-mortality . The comp = 0.0 06 and β = 0.509, p < 0.0001, respectively). Age at KT was also associated with the worsening of arterial stiffness in the late post-transplantation period, even after adjusting for PWV at 1 year ). A higher PWV at 1-year was associated with a PWV decrease over the late post-transplantation period   . ImportaDespite the improvement of renal function conferred by KT, CV mortality remains higher in KT recipients compared with the general population. In KT, some studies demonstrated that an increased arterial stiffness was associated with CV mortality ,4. NeverThe present study focuses on the association between CV morbi-mortality and the PWV measured at different time-points, as well as the evolution of the PWV through time. Notably, despite the low number of patients, we had, for the first time, the opportunity to measure the arterial stiffness in the pre-KT, the early post-KT, and the late post-KT periods. Moreover, we analyzed CV outcomes after a ten-year follow-up period.In a study on 220 kidney transplant recipients, carotid–femoral PWV evaluated at 3 and 12 months after KT was significantly associated with morbi-mortality 5.5 years after KT, whereas the delta of PWV between month 12 and month 3 was not . HoweverUnsurprisingly, age is associated with increased PWV at 1 year, but also with less improvement in the long-term post-transplantation period. Ignace et al. previously reported that older patients had a higher improvement in PWV in the early post-transplantation period at 3 months post-KT . We can On the contrary, increased PWV at 1 year is associated with increased potential improvement in the long-term post-transplantation period. One can hypothesize that arterial stiffness mainly related to CKD, but not age, has a greater potential for recovery after KT, and that the susceptibility of the vascular system to immunosuppressive therapy is less pronounced among the youngest patients with ESRD.It is worth mentioning that, similarly to the results of Kolonko et al. , a histoThis study is a single-center study with a small sample size. This prevented the use of more advanced statistical analyses such as splines to determine the most adequate prognostic PWV thresholds, or the addition of some possibly important covariates in the multivariable analysis. However, we assessed ESRD-related arterial stiffness over a long period of time in a peculiar population of kidney-transplanted patients, something that has not been reported previously. Further long-term studies should be conducted to better ascertain the long-term changes in PWV following KT.There was no preemptive KT in this cohort. Therefore, it was not possible to include the residual renal function before KT to assess its potential impact on the evolution of arterial stiffness after KT.In this analysis, the absence of PWV- worsening in the late post-transplantation period was significantly associated with a lower risk of CV events. In contrast, the change in PWV observed early during the post-transplantation period was not associated with CV events.In summary, the present study suggests that the worsening of aortic stiffness in the late post-KT period has a negative impact on long-term CV morbi-mortality. Ultimately, finding an intervention able to reduce PWV could improve the prognosis of KT recipients.


Bio-C Pulpecto (Bio-CP) was recently developed as the first bioceramic root filling material for primary teeth.  To evaluate the physicochemical properties of radiopacity, setting time, pH, cytocompatibility and potential of Bio-CP to induce mineralisation, compared with (1) Calen thickened with zinc oxide , and (2) zinc oxide and eugenol (ZOE). Physicochemical properties were evaluated according to ISO 6876. Saos-2 (human osteoblast-like cell line) exposed to extracts of the materials were subjected to assays of methyl thiazolyl tetrazolium, neutral red, alkaline phosphatase (ALP) activity and mineralised nodule production. The results were analysed using one-way or two-way ANOVA and Tukey’s or Bonferroni’s post-tests (α=0.05). All the materials showed radiopacity higher than 3 mm Al. Bio-CP had lower pH than Calen-ZO, but higher pH than ZOE. Calen-ZO and Bio-CP did not set. The setting time for ZOE was 110 min. The cytocompatibility order was Calen-ZO > Bio-CP > ZOE  and Calen-ZO > Bio-CP = ZOE  and Calen-ZO = Bio-CP > ZOE (1:32 dilution). Bio-CP induced greater ALP activity at 7 days, and greater mineralised nodule production, compared to Calen-ZO (p<0.05). Conclusions Bio-CP showed adequate physicochemical properties, cytocompatibility and potential to induce mineralisation. It is composed of ester glycol salicylate, titanium oxide, calcium tungstate, silicon dioxide, toluene sulphonamide, and calcium silicate . According to its manufacturer, it presents high alkalinity (pH 12.7), high radiopacity , complies with ISO 6876,15 and is resorbable, thus allowing simultaneous physiological resorption of the root and the material . A recent study showed that the Bio-CP is biocompatible and induced biomineralization in the subcutaneous tissue of rats, in a manner similar to MTA.16 In addition, a root filling material for primary teeth developed by Angelus, with similar composition of Bio-CP, but with the name not informed, showed good biocompatibility in the subcutaneous tissue of rats, low cytotoxicity in human gingival fibroblast, and satisfactory behaviour regarding the physicochemical properties studied.8 However, the scientific literature about Bio-CP is still scarce.New endodontic cements have been developed, especially highlighting the advance in improved bioceramics. The main characteristics of bioceramic materials for endodontic use are their alkaline pH, shrink-free property, chemical stability within the biological environment,in vitro assays. The null hypothesis was that there would be no difference in the physicochemical and biological properties among the materials.Thus, the aim of this study was to evaluate (1) the physicochemical properties of radiopacity, setting time and pH, and (2) the biological properties of cytocompatibility and potential to induce mineralisation of Bio-CP, compared with Calen-ZO and ZOE, Root filling materials, manufacturers and proportions for manipulation are shown in 15 .Eight specimens of each material, measuring 10 mm diameter and 1 mm high were prepared according to ISO 6876:201215 . The speTen polyethylene tubes 10-mm long with 1 mm internal diameter were filled with each material. Each specimen was immersed in 10 mL of deionised water, inside of a plastic flask sealed individually with a lid, and then stored at 37°C. The experimental time intervals were 1, 3, 7, 14 and 28 days. The pH of the solution was assessed with a previously calibrated digital pH meter  at each time interval. The control group consisted of deionised water with no immersed material.15 The setting time of the specimens was established as the time elapsing from initial mixing of Calen-ZO and syringe application of Bio-CP up to the maximum hardening of the specimens. At this point, the indentation marks made by the Gillmore needle were substantially reduced, but not eliminated. This is because the pastes had not set. Comparatively, the setting time for ZOE was considered as the time elapsing from when it started to be mixed until such time as the Gillmore needle failed to leave marks on the surface of the specimens.Calen-ZO and ZOE were inserted into ring-shaped metal moulds 1 mm wide and 10 mm diameter (n=6), kept at 37°C and 95% humidity. A plaster mould with the same dimensions was prepared for Bio-CP. The plaster mould was previously submerged in deionised water for 24 hours to provide the bioceramic material with moisture. The setting time was assessed using a Gillmore needle with a mass of 100 g ±0.5 and diameter of 2.0 mm ±0.1, according to ISO 6876:2012.Saos-2 (human osteoblast-like cell) immortalized cells line were grown in flasks containing Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , penicillin (100 IU / mL), and streptomycin (100 μg / mL), in an atmosphere with 5% CO2 and 95% humidity at 37°C.18 Afterwards, the supernatant was transferred to new microtubes centrifuged for 10 min at 20,800 g  to decant any particles of material left in the supernatant. The supernatant was transferred to a new tube and was considered as the “stock solution/extract”. It was then diluted and placed in contact with Saos-2 cells.19After handling of the specimens, 100 mg of each material  was plac5 cells/mL in a 96-well plate containing DMEM with 10% SFB for 24 hours to adhere to the plates. Thereafter, the cells were exposed to the cement extracts at the following dilutions in DMEM (v: v) 1:2; 1:4; 1:12; 1:24; 1:32. Cells exposed to DMEM were considered as the control group.Saos-2 cells were cultured at a density of 1x102 for 3 hours. The colorimetric product was solubilised in 100 µl of 0.04 N acidified isopropanol (Sigma-Aldrich). A spectrophotometer  at 570 nm was used to measure the optical densities of the solutions. Absorbance readings were normalised to readings of cells exposed to DMEM, and represented the succinate dehydrogenase activity (cellular metabolism).After 24 hours of cell contact with the material extracts and the control, 100 μL of 5 mg / mL MTT solution (Sigma-Aldrich) was added to each well, followed by incubation at 37°C, 95% humidity and 5% CO2 for 3 hours, the contents of the wells were removed, and the colorimetric product was solubilised with 100 μL of a solution containing 50% ethanol and 1% acetic acid (Sigma-Aldrich). A spectrophotometer  at 570 nm was used to measure the optical densities of the solutions. Absorbance readings were normalised to readings of cells exposed to DMEM, and represented the ability to incorporate the dye into viable cell lysosomes.The NR assay was performed by applying 100 μL DMEM containing NR at 50µg / mL (Sigma-Aldrich) after 24 hours of cell contact with the materials and control extracts. The cells were incubated at 37°C, 95% humidity and 5% CO4 cells / mL) were cultivated in 96-well plates and exposed to the control (DMEM) and the extract of the materials at 1:24 dilution. This dilution was selected after observing the results of MTT assays performed after exposure of cells to cement extracts for 1, 3 and 7 days (data not shown). The 1:24 dilution was the highest extract concentration without cytotoxic effects. This is an essential consideration, since dead cells do not show ALP activity. ALP activity was evaluated at periods of 1, 3 and 7 days, using the commercial kit . Extracts of the materials were renewed every two days. Cells were washed with 200 μL saline phosphate buffer (PBS) after each experimental period, and a 200 μL sodium sulphate solution  was added to each well. Then the samples were left to stand for 30 minutes at room temperature. Each sample (5 μL) in the sodium sulphate solution was transferred to a microtube (Eppendorf) containing a substrate and a buffer enzyme. Absorbance was measured using a spectrophotometer at 590 nm. Data were expressed as normalised ALP activity with total protein content, detected using the Total Protein Kit (Labtest) at the respective experimental periods. MTT assays were performed together with the ALP activity assay to follow up cell viability.Saos-2 (5x104 cells / mL) in 12-well culture plates with DMEM, supplemented with 50 µg / mL L-ascorbic acid (Sigma-Aldrich) and 10 mM β glycerophosphate (Sigma-Aldrich). The cells were exposed to material extracts for 21 days at the same dilution used for the ALP activity assay. Material extracts were renewed every two days. Then the cells were fixed with 4% paraformaldehyde (Sigma) and stained with 2% ARS (pH 4.1). The cells were incubated at room temperature for 20 minutes, and the dye was aspirated. The wells were washed 4 times with 1 mL of distilled water for 5 minutes. The water was removed and mineralisation was quantified by dissolving the nodules with 1 mL of a 10% cetylpyridinium chloride solution (Sigma Aldrich) added to each well, after which the plate was incubated for 15 minutes under stirring at room temperature. Three 100 μL aliquots of the suspension from each well were transferred to a 96-well plate and read with a 562 nm wavelength filter on a spectrometer .Saos-2 cells were cultivated  and the Tukey’s post-test, or two-way ANOVA and Bonferroni’s post-test (α=0.05), using GraphPad Prism statistical software .15 There was no difference between Bio-CP and Calen-ZO (p>0.05), and ZOE showed higher radiopacity than the other materials (p<0.05). The ZOE setting time was 110 minutes. Calen-ZO and Bio-CP were considered to “set” (maximum hardening) at 192 hours and 240 hours, respectively. Although Calen-ZO and Bio-CP pastes had alkaline pH, Calen-ZO had a higher pH than Bio-CP (p<0.05). ZOE had a neutral pH  was less than that stated by the manufacturer (9 mm AL). On the other hand, it agrees with a previous study that showed that an experimental MTA-based material, with similar composition of Bio-CP, had radiopacity of 3.28.8 The greater radiopacity of ZOE (9.25 mm Al) than that of the other materials (3.5 mm Al) is attributed to its zinc oxide used as a radiopacifier.21Radiopacity is an essential property for endodontic materials, because it allows endodontic filling material to be viewed by radiographic examination to evaluate the obturation quality,22 Both Bio-CP and Calen-ZO had alkaline pH; however, Calen-ZO had a higher alkaline pH compared with Bio-CP over the evaluated periods. Calcium hydroxide dissociates into calcium and hydroxyl ions, which raises the pH of the medium. Bioceramic materials form calcium hydroxide when they undergo a hydration reaction.12The lower pH of Bio-C compared to Calen-ZO is probably due to the lower amount of calcium hydroxide released into the medium by Bio-CP.An alkaline medium not only neutralises lactic acid from osteoclasts, but also prevents the mineral components of dentine from dissolving. In addition, it promotes antimicrobial activity, and may activate alkaline phosphatases, which play an important role in hard-tissue formation.23 Because Bio-CP was developed as a root filling material for primary teeth, is desirable that the material can be reabsorbed to accompany the physiological resorption of deciduous teeth roots. It can be hypothesized that the absence of set, along with the composition and solubility of the material, can allow simultaneous physiological resorption of the root and the material.ZOE had a setting time of 110 minutes. The hardening time of Calen-ZO was 192 hours and that of Bio-CP was 240 hours; neither material set. The fact that Bio-CP does not set is not a disadvantage, since this phenomenon is observed in materials considered suitable for filling root canals of primary teeth.17 MTT and NR assays were used to assess the cytocompatibility of the materials. MTT is based on the ability of viable cells to metabolise the MTT salt. NR assesses the ability of viable cells to incorporate dye into lysosomes.17 Studies that evaluate different cell parameters make the cytocompatibility results more reliable24 ALP activity and mineral nodule production assays were performed to evaluate the potential of materials to induce mineralisation. ALP activity assay is a biomarker of the enzyme alkaline phosphatase produced by osteoblasts in their early maturation process,25 and ARS identifies calcium deposits in cell culture.17Saos-2 cells were used because they provide a suitable model to evaluate cytocompatibility and the potential to induce mineralisation of the materials.26 human gingival fibroblasts27and murine osteoblast precursor cells (IDG- SW3).13 Moreover this material promoted osteoblastic differentiation,13 showed anti-inflammatory effects and induced mineralisation in osteoblast precursor cells (MC3T3-E1).14 Another study comparing mineral trioxide aggregate – MTA  and iRoot SP  showed that neither material was cytotoxic in human tooth stem cells.28The cytocompatibility and the potential to induce mineralisation of bioceramic endodontic materials for permanent teeth has been studied in different cell types. EndoSequence BC sealer  showed low cytotoxicity in L929 fibroblasts,8 (2017) showed no statistical difference in the cytotoxicity of Calen-ZO and the experimental MTA-based material with a similar composition of Bio-CP. This difference in result can be explained by the type of cells and also by the Calen-ZO proportion used. While in the present study Saos-2 cells (osteoblast-like cells), and Calen-ZO in a 2: 1 proportion were used, Pilownic, et al. 8 (2017) used human gingival fibroblasts and Calen-ZO in a 1: 1 proportion. It is important to emphasize that it is not clear if the manufacturer modified the concentrations of the components in Bio-CP, because at the time of the Pilownic, et al.8 (2017) study, the material did not have a name yet and, unfortunately, the manufacturer did not disclose the concentrations.Bio-CP cytocompatibility was lower compared to Calen-ZO, but higher compared to ZOE. Pilownic, et al.16 Regarding Calen-ZO, the results of the present study are in line with previous studies performed on dogs’ teeth and the subcutaneous tissue of mice, which showed that Calen-Zo was more biocompatible than ZOE.10 ZOE was not evaluated in either mineralisation assay conducted in the present study, because it has no bioactivity.29The high mineralisation potential of calcium-silicate-based bioceramic materials is attributed to their hydration, that is, a chemical reaction with tissue fluids yields hydrated silicate gel (C-S-H) and calcium hydroxide.30 These compounds  each have a mineralisation pathway. Calcium hydroxide induces the formation of calcite crystals that induce the formation of calcified areas. Calcite crystals are formed from the dissociation of calcium ions from calcium hydroxide, and carbon dioxide ions from tissues.31 On the other hand, C-S-H induces hydroxyapatite deposition; this reaction is promoted by the hydration of tissue fluids. In bioceramic materials, calcium hydroxide reacts with the phosphate ions in tissue fluids to deposit calcium phosphate.30Bio-CP induced lower ALP activity in 1 day, but greater ALP activity in 7 days, than Calen-ZO. In addition, Bio-CP induced greater mineralised nodule production than Calen-ZO. Calen-ZO induced higher calcium deposits than the control group. A recent study showed that Bio-CP was biocompatible and induced biomineralization similar to MTA in the subcutaneous tissue of rats.in vivo studies. In addition, to complement the research on Bio-CP, studies should be carried out to evaluate its antibiofilm activity and the capacity to be reabsorbed to accompany the physiological resorption of deciduous teeth roots.The results obtained for cytocompatibility and mineralization induction should be confirmed by further It was concluded that Bio-CP did not set, presented adequate radiopacity and alkaline pH, was cytocompatible and had the potential to induce mineralisation. Considering its good physicochemical and biological properties, Bio-CP has the potential to become an adequate material for the root canal filling of primary teeth.


Various circuit techniques are exploited in this work, such as the feedforward transimpedance amplifier for high gain, and a limiting amplifier with negative impedance compensation for wide bandwidth. Measured results demonstrate 93.4-dBΩ transimpedance gain, 790-MHz bandwidth, 12-pA/√Hz noise current spectral density, 6.74-μApp minimum detectable signal that corresponds to the maximum detection range of 10 m, and 56.5-mW power dissipation from a 1.8-V supply. This optoelectronic Rx IC provides a potential for a low-cost low-power solution in the applications of home-monitoring LiDAR sensors.This paper presents an optoelectronic receiver (Rx) IC with an on-chip avalanche photodiode (APD) realized in a 0.18-μm CMOS process for the applications of home-monitoring light detection and ranging (LiDAR) sensors, where the on-chip CMOS P Light detection and ranging (LiDAR) sensors have been proliferated for the past decade because they can be applied to diverse fields such as 3-dimensional imaging for unmanned self-driving cars, and medical and industrial applications . ParticuIn these LiDAR sensors, avalanche photodiodes (APDs) are mostly exploited as an off-chip optical detector. However, these off-chip APD devices may increase the packaging cost considerably in the cases of multi-channel Rx arrays and deteriorate signal integrity because of the bondwires between APDs and receiver (Rx) chips. Furthermore, on-chip electro-static discharge (ESD) protection diodes may be needed, hence shrinking the Rx bandwidth by the increased input capacitance. Therefore, we present an on-chip CMOS APD in this work which can be a better solution to resolve the aforementioned issues.Yet, on-chip CMOS APDs suffer from low responsivity and limited bandwidth when compared to the discrete APD devices. Especially, silicon CMOS implementation of on-chip APDs restricts their operations only in the wavelength of 850 nm . NonetheThe target application of this optoelectronic Rx IC is home-monitoring LiDAR sensors for both single elders who live alone and senile dementia patients that reside in either hospitals or nursing homes. Therefore, the optoelectronic LiDAR sensors mandate the following specifications. First, it should be able to detect the distance within 10 m. Second, owing to the lack of responsivity, avalanche multiplication should be equipped in on-chip CMOS photodiodes. Third, the generated photocurrents from on-chip CMOS APDs may be still in the range of a few micro-amperes even with the avalanche multiplication. Therefore, the front-end TIA circuit should provide high transimpedance gain so as to recover the weak incoming signals. In other words, the LiDAR sensors should be able to lower the minimum detectable signal (MDS).+/N-well architecture that can yield large avalanche gain owing to the guard ring structure of shallow trench isolation (STI). The avalanche multiplication occurs in the P+/N-well junction. It should be noted that the P+ contacts in the N-well are connected to the input node of the following TIA circuit, hence excluding the slow diffusion currents contributed from the P-substrate.+/N-well junction, so that the STI can prevent premature edge breakdown. Furthermore, the high electric-field can be distributed uniformly at the planar junction [The STI guard rings are designed to be deeper in depth than the Pjunction .+/N-well APD and its chip microphotograph, where the shape is octagonal to minimize the possible damage from edge breakdown. The P+ source/drain region is covered by the salicide blocking layer to form an optical window. However, the P+ contacts located in the middle of the optical window cannot be blocked because the salicide process reduces the contact resistivity [istivity .+ contacts should be small enough not to deteriorate the APD responsivity. In this work, the diagonal length of the optical window is designed to be 40 µm.Area occupied by the P+/N-well APD, a feedforward TIA, a three-stage LA with negative impedance compensation, and an OB.The front-end feedforward TIA determines the overall performance of the optoelectronic Rx IC, therefore mandating stringent requirements of high transimpedance gain to recover weak incoming signals, low noise current spectral density to minimize the MDS, differential signaling for common-mode noise rejection, etc.FR), a feedforward common-source amplifier with its gate connected to the gates of the INV stage, a differential pair with a low-pass-filter for single-to-differential conversion, and an additional differential gain stage for gain boosting further.mi(i=1 to 3)g and oi(i=1 to 3)r represent the transconductance and the output resistance of each transistor.According to the small signal analysis of the simplified feedforward input stage (as described in ), the trk is the Boltzmann’s constant, T is the absolute temperature, and T C represents the total capacitance at the input node of the feedforward TIA which includes the photodiode capacitance (DC) and the input capacitance of the INV input stage, i.e., in,M1 + Cin,M2 = Cgs1 + Cgs2 + (1 + Av)·(Cgd1 + Cgd2)C. It is noted that GR is set to 1 kΩ in this work so that the noise contribution from RG can be negligible.The input-referred noise current spectral density of the simplified feedforward input stage (as described in ) is giveBWn1 is the noise bandwidth for white noise and BWn2 is the noise bandwidth for f2 noise. For Q = 1/1nBW ≈ 1.11·33dBBW and 2nBW ≈ 1.49·3dBBW [Then, the input-referred mean-square noise current is given by,49·BW3dB .LR is selected to a few tens of kilo-ohm. Then, the bias current (I2) of M3 can be mostly supplied through the PMOS (M2) of the INV stage, because the DC drain voltage of M2 is fixed by the action of the INV stage via the feedback resistor (FR). Hence, this action enables to boost the transconductance (m2g) of M2 and helps to reduce the noise current spectral density of the feedforward TIA.In rms.Post-layout simulations were conducted by utilizing the model parameters of a standard 0.18-µm CMOS process. Limiting amplifier (LA) amplifies the small output voltage signals of the feedforward TIA to sufficient levels so that a following TDC can detect the signals precisely. Furthermore, the bandwidth of LA should be wide enough not to degrade the operating speed of the preceding TIA. The schematic diagram of a gain-cell is shown in mg represents the transconductance of transistors in the negative impedance circuits and gsC represents the gate-source capacitance of transistors in the negative capacitance circuit.Small signal analysis shows that the equivalent resistance and capacitance are given by,fA, consequently removing the offset voltage.Moreover, a DC offset network should be employed because an input offset voltage, however tiny it may be, can be amplified to saturate the output of the LA. fA, a low cutoff-frequency is newly introduced at v,LAA is the voltage gain of the LA [f should be judiciously selected to a reasonable value so that the feedback network maintains the circuit stability.Even though the input offset voltage is suppressed by f the LA . Therefo2. DC measurements reveal that the optoelectronic Rx IC consumes 56.5 mW from a single 1.8-V supply.Test chips of the proposed optoelectronic Rx IC were fabricated in a standard 0.18-μm CMOS process. +/N-well APD under the conditions of both dark and optical illuminations. It is clearly seen that the dark current and the illumination current rise sharply at the breakdown voltage of 11.1 V owing to the avalanche multiplication process, where the incident optical power is about –60 dBm. The responsivity (R) of the on-chip CMOS P+/N-well APD is then given by,illuminationI and darkI represent the measured currents under illumination and dark conditions, respectively. Popt represents the incident optical power. 21 of 26 dB are achieved with a 50-Ω termination. It leads to the single-ended transimpedance gain (Z21) of 87.4 dBΩ by the following equation,rms is then translated to 6.74-µApp MDS since the signal-to-noise ratio (SNR) of 10 is required for successful detection [This measured input-referred RMS noise of 337-nAetection . With APpp 231-1 pseudo random bit sequence (PRBS) inputs at different operation speeds of 100 Mb/s and 500 Mb/s, respectively. Measurements confirm that the proposed optoelectronic Rx IC achieves wide and clean eyes up to 800-Mb/s operations.+/N-well APD, clearly showing the final output voltage pulses.This work is the first attempt to integrate an on-chip CMOS APD with analog frontend IC for the applications of LiDAR sensors. Despite the well-known inferior performance of an on-chip CMOS APD, the proposed optoelectronic Rx IC provides comparable performance with a little expense of noise degradation. Yet, it would barely be a critical issue for home-monitoring sensors with 10-m detection range.+/N-well APD demonstrates the responsivity of 2.72 A/W at the reverse bias voltage of 11.05 V, and the whole optoelectronic Rx IC achieves the differential transimpedance gain of 93.4 dBΩ, bandwidth of 790 MHz, and maximum detection range of 10 m with power consumption of 56.5 mW. It can be concluded that this work certainly provides a low-cost solution for short-range indoor LiDAR sensors.The first CMOS optoelectronic Rx IC was realized for the applications of indoor home-monitoring LiDAR sensors. The on-chip P